Methodological Pipeline Research Articles

An End-to-End DNA Taxonomy Methodology for Benthic Biodiversity Survey in the Clarion-Clipperton Zone, Central Pacific Abyss

Recent years have seen increased survey and sampling expeditions to the Clarion-Clipperton Zone (CCZ), central Pacific Ocean abyss, driven by commercial interests from contractors in the potential extraction of polymetallic nodules in the region. Part of the International Seabed Authority (ISA) regulatory requirements are that these contractors undertake environmental research expeditions to their CCZ exploration claims following guidelines approved by the ISA Legal and Technical Commission (ISA, 2010). Section 9 (e) of these guidelines instructs contractors to “…collect data on the sea floor communities specifically relating to megafauna, macrofauna, meiofauna, microfauna, nodule fauna and demersal scavengers”. There are a number of methodological challenges to this, including the water depth (4000–5000 m), extremely warm surface waters (~28 °C) compared to bottom water (~1.5 °C) and great distances to ports requiring a large and long seagoing expedition with only a limited number of scientists. Both scientists and regulators have recently realized that a major gap in our knowledge of the region is the fundamental taxonomy of the animals that live there; this is essential to inform our knowledge of the biogeography, natural history and ultimately our stewardship of the region. Recognising this, the ISA is currently sponsoring a series of taxonomic workshops on the CCZ fauna and to assist in this process we present here a series of methodological pipelines for DNA taxonomy (incorporating both molecular and morphological data) of the macrofauna and megafauna from the CCZ benthic habitat in the recent ABYSSLINE cruise program to the UK-1 exploration claim. A major problem on recent CCZ cruises has been the collection of high-quality samples suitable for both morphology and DNA taxonomy, coupled with a workflow that ensures these data are made available. The DNA sequencing techniques themselves are relatively standard, once good samples have been obtained. The key to quality taxonomic work on macrofaunal animals from the tropical abyss is careful extraction of the animals (in cold, filtered seawater), microscopic observation and preservation of live specimens, from a variety of sampling devices by experienced zoologists at sea. Essential to the long-term iterative building of taxonomic knowledge from the CCZ is an “end-to-end” methodology to the taxonomic science that takes into account careful sampling design, at-sea taxonomic identification and fixation, post-cruise laboratory work with both DNA and morphology and finally a careful sample and data management pipeline that results in specimens and data in accessible open museum collections and online repositories.

Identifying miRNA-mediated signaling subpathways by integrating paired miRNA/mRNA expression data with pathway topology.

In the road for network medicine the newly emerged systems-level subpathway-based analysis methods offer new disease genes, drug targets and network-based biomarkers. In parallel, paired miRNA/mRNA expression data enable simultaneously monitoring of the micronome effect upon the signaling pathways. Towards this orientation, we present a methodological pipeline for the identification of differentially expressed subpathways along with their miRNA regulators by using KEGG signaling pathway maps, miRNA-target interactions and expression profiles from paired miRNA/mRNA experiments. Our pipeline offered new biological insights on a real application of paired miRNA/mRNA expression profiles with respect to the dynamic changes from colostrum to mature milk whey; several literature supported genes and miRNAs were recontextualized through miRNA-mediated differentially expressed subpathways.

Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing.

BK virus (BKV) infection causing end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep-sequencing methodology and bioinformatics pipeline that identify BKV variants across the genome and at BKV-specific HLA-A2-, HLA-B0702-, and HLA-B08-restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets, and fragmentation libraries were sequenced on the Ion Torrent Personal Genome Machine (PGM). An error model and variant-calling algorithm were developed to accurately identify rare variants. A total of 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range, 2 to 37; interquartile range, 10), with the majority of variants (77%) detected at a frequency of <5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for the BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies.

Dynamic transcriptional signatures and network responses for clinical symptoms in influenza-infected human subjects using systems biology approaches.

Recent studies demonstrate that human blood transcriptional signatures may be used to support diagnosis and clinical decisions for acute respiratory viral infections such as influenza. In this article, we propose to use a newly developed systems biology approach for time course gene expression data to identify significant dynamically response genes and dynamic gene network responses to viral infection. We illustrate the methodological pipeline by reanalyzing the time course gene expression data from a study with healthy human subjects challenged by live influenza virus. We observed clear differences in the number of significant dynamic response genes (DRGs) between the symptomatic and asymptomatic subjects and also identified DRG signatures for symptomatic subjects with influenza infection. The 505 common DRGs shared by the symptomatic subjects have high consistency with the signature genes for predicting viral infection identified in previous works. The temporal response patterns and network response features were carefully analyzed and investigated.

Combining EEG and MEG for the Reconstruction of Epileptic Activity Using a Calibrated Realistic Volume Conductor Model

To increase the reliability for the non-invasive determination of the irritative zone in presurgical epilepsy diagnosis, we introduce here a new experimental and methodological source analysis pipeline that combines the complementary information in EEG and MEG, and apply it to data from a patient, suffering from refractory focal epilepsy. Skull conductivity parameters in a six compartment finite element head model with brain anisotropy, constructed from individual MRI data, are estimated in a calibration procedure using somatosensory evoked potential (SEP) and field (SEF) data. These data are measured in a single run before acquisition of further runs of spontaneous epileptic activity. Our results show that even for single interictal spikes, volume conduction effects dominate over noise and need to be taken into account for accurate source analysis. While cerebrospinal fluid and brain anisotropy influence both modalities, only EEG is sensitive to skull conductivity and conductivity calibration significantly reduces the difference in especially depth localization of both modalities, emphasizing its importance for combining EEG and MEG source analysis. On the other hand, localization differences which are due to the distinct sensitivity profiles of EEG and MEG persist. In case of a moderate error in skull conductivity, combined source analysis results can still profit from the different sensitivity profiles of EEG and MEG to accurately determine location, orientation and strength of the underlying sources. On the other side, significant errors in skull modeling are reflected in EEG reconstruction errors and could reduce the goodness of fit to combined datasets. For combined EEG and MEG source analysis, we therefore recommend calibrating skull conductivity using additionally acquired SEP/SEF data.

Robust 4C-seq data analysis to screen for regulatory DNA interactions

Regulatory DNA elements can control the expression of distant genes via physical interactions. Here we present a cost-effective methodology and computational analysis pipeline for robust characterization of the physical organization around selected promoters and other functional elements using chromosome conformation capture combined with high-throughput sequencing (4C-seq). Our approach can be multiplexed and routinely integrated with other functional genomics assays to facilitate physical characterization of gene regulation.

A solid quality-control analysis of AB SOLiD short-read sequencing data

Next generation sequencers have greatly improved our ability to mine polymorphisms and mutations out of entire (or portions of) genomes. The reliability of their outputs, though, showed to be very related to the sequencing chemistry and to deeply affect the quality of the downstream analyses. We focus here on the two-base color code chemistry of AB SOLiD sequencers and propose a comprehensive quality control methodological and software pipeline. We used existing and custom tools to detect and purge short-reads of some common flaws due to sequencing errors and chemical hitches. We apply them to a cohort of SOLiD 4 runs and measure their joint efficacy in terms of the resulting ability to detect the greatest possible number of true variants.

Sleeping Beauty transposon mutagenesis in rat spermatogonial stem cells

We describe an experimental approach for generating mutant alleles in rat spermatogonial stem cells (SSCs) using Sleeping Beauty (SB) transposon-mediated insertional mutagenesis. The protocol is based on mobilization of mutagenic gene-trap transposons from transfected plasmid vectors into the genomes of cultured stem cells. Cells with transposon insertions in expressed genes are selected on the basis of activation of an antibiotic-resistance gene encoded by the transposon. These gene-trap clones are transplanted into the testes of recipient males (either as monoclonal or polyclonal libraries); crossing of these founders with wild-type females allows the insertions to be passed to F(1) progeny. This simple, economic and user-friendly methodological pipeline enables screens for functional gene annotation in the rat, with applicability in other vertebrate models where germ line-competent stem cells have been established. The complete protocol from transfection of SSCs to the genotyping of heterozygous F(1) offspring that harbor genomic SB gene-trap insertions takes 5-6 months.

Investigating the Minimum Required Number of Genes for the Classification of Neuromuscular Disease Microarray Data

The discovery of potential microarray markers, which will expedite molecular diagnosis/prognosis and provide reliable results to clinical decision-making and treatment selection for patients, is of paramount importance. Feature selection techniques, which aim at minimizing the dimensionality of the microarray data by keeping the most statistically significant genes, are a powerful approach toward this goal. In this paper, we investigate the minimum required subsets of genes, which best classify neuromuscular disease data. For this purpose, we implemented a methodology pipeline that facilitated the use of multiple feature selection methods and subsequent performance of data classification. Five feature selection methods on datasets from ten different neuromuscular diseases were utilized. Our findings reveal subsets of very small number of genes, which can successfully classify normal/disease samples. Interestingly, we observe that similar classification results may be obtained from different subsets of genes. The proposed methodology can expedite the identification of small gene subsets with high-classification accuracy that could ultimately be used in the genetics clinics for diagnostic, prognostic, and pharmacogenomic purposes.

Towards a molecular phylogeny of Mollusks: Bivalves’ early evolution as revealed by mitochondrial genes

Despite huge fossil, morphological and molecular data, bivalves’ early evolutionary history is still a matter of debate: recently, established phylogeny has been mostly challenged by DNA studies, and little agreement has been reached in literature, because of a substantial lack of widely-accepted methodological approaches to retrieve and analyze bivalves’ molecular data. Here we present a molecular phylogeny of the class based on four mitochondrial genes (12s, 16s, cox1, cytb) and a methodological pipeline that proved to be useful to obtain robust results. Actually, best-performing taxon sampling and alignment strategies were tested, and several data partitioning and molecular evolution models were analyzed, thus demonstrating the utility of Bayesian inference and the importance of molding and implementing non-trivial evolutionary models. Therefore, our analysis allowed to target many taxonomic questions of Bivalvia, and to obtain a complete time calibration of the tree depicting bivalves’ earlier natural history main events, which mostly dated in the late Cambrian.

Moving pieces in a taxonomic puzzle: Venom 2D-LC/MS and data clustering analyses to infer phylogenetic relationships in some scorpions from the Buthidae family (Scorpiones)

The Buthidae is the most clinically important scorpion family, with over 500 species distributed worldwide. Taxonomical positions and phylogenetic relationships concerning the representative genera and species of this family have been mostly inferred based upon comparisons between morphological characters. Yet, some authors have performed such inferences by comparing some structural properties of a few selected molecules found in the venoms from these scorpions. Here, we propose a novel methodology pipeline designed to address these issues. We have analyzed the whole venoms from some species that exemplify peculiar cases in the Buthidae family ( Tityus stigmurus, Tityus serrulatus, Tityus bahiensis, Leiurus quinquestriatus quinquestriatus and Leiurus quinquestriatus hebraeus), by means of a proteomic approach using a 2D-LC/MS technique. The molecules found in these venoms were clustered according to their physicochemical properties (molecular mass and hydrophobicity), by using the machine learning-based Weka software. The clusters assessment, along with the number of molecules found in a given cluster for each scorpion, which assigns for the venom and structural family complexities, respectively, was used to generate a phenetic correlation tree for positioning these species. Our results were in accordance with the classical taxonomy viewpoint, which places T. serrulatus and T. stigmurus as very close species, T. bahiensis as a less related species in the Tityus genus and L. q. quinquestriatus and L. q. hebraeus with small differences within the same species ( L. quinquestriatus). Therefore, we believe that this is a well-suited method to determine venom complexities that reflect the scorpions' evolutionary history, which can be crucial to reconstruct their phylogeny through the molecular evolution of their venoms.

Fracture control assessment in the base metal and welded joint of a pipeline steel

ABSTRACTThe present study evaluates the fracture behaviour of a pipe steel and compares the real scale pipeline assessment methodology (pipeline methodology) with the Guidance on Methods for Assessing the Acceptability of Flaws in Welded Structures (PD 6493). It is shown that the base metal is the toughest region and that the heat‐affected zone is the least tough zone, that both assessment methods do not give the same result for the base metal, and that plasticity at the crack tip can toughen the welded joint.